CRISPR Tools

CRISPR Tools

CRISPR analysis toolsgRNA design tools and off-target prediction:First you have to design guide RNA (gRNA) recognition sites in you target sequence. The target has to be flanked by a PAM site (which is NGG in the case of the most widely used S. pyogenes Cas9). You want to design the gRNA as specific as you can, with limited homology to other regions in the genome. Please find below some online design tools that help you design the best gRNAs. Note however that the off-target predictions are very imprecise. You might want to run your gRNA on the CROP-IT, which is one of the best performing off-target identification software.

MIT, Feng Zhang lab CRISPR design toolAvailable for gRNA design, nickase analysis and off-target prediction (for 15 different species). Gives the user a score based on the number of mismatches and the position of mismatches in the gRNA (PAM proximal binding is more stringent). Gives a quality assessment of each gRNAs based on homology to off-target sites.

E-CRISPR, Michael Boutros lab CRISPR design toolIncludes plenty of advanced design options. The user can select from 33 organisms for off-target search. Input is a user-defined target gene, either in FASTA format or gene symbol. User can define the purpose of application. Can search for NGG and NAG PAM sites as well. It is very useful that different regions of genes (5' part, 3' part, exons, avoids CpG islands etc.) can be specified in the input. Scores gRNAs based on efficacy, annotation and specificity.

CROP-IT, CRISPR design and off-target analysis toolThe CRISPR design only allows 250bp sequences. The off-target analysis is very useful, it separates off-targets between cleavage and Cas9 binding (e.g. epigenome editing requires only binding of dCas9), since cleavage and binding have different tolerance to mismatches.

ChopChop CRISPR design, HarvardAllows for gRNA and TALEN recognition sites, recognition sequences are visualized along the gene of interest, off-target scoring. Incorporates some Cas9 variants. A useful feature is that it gives primer sequences for amplification.

Cloning guides and resourcesNext, you have to clone your gRNA into one of the available backbones. Please find below some well-known Cas9 plasmids and respective cloning guides. Most of them are available through Addgene (click on the name to access it). A very useful guide is available through Addgene.

CRISPR for transcriptional regulationActivating proteins are usually based on dCas9-VP64 or BFP fusion proteins (d stands for dead, catalytically inactive Cas9). Inhibiting proteins are usually based on dCas9-KRAB fusion proteins.

CRISPR librariesYou can use a pool of different gRNAs to multiplex the CRISPR system. Here are some available libraries:

Jonathan Weissman libraries (CRISPRa and CRISPRi) for human cellsCRISPR is used to activate (CRISPRa) or inhibit (CRISPRi) gene expression. It can influence the transcription of 15,977 different genes. For CRISPRa, use the dCas9-VP64, for CRISPRi use thedCas9-KRAB.